MATERIALS AND METHODS

Cell Cultures and Reagents Normal human mammary epithelial MCF-10A cells (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 medium supplemented with 5% horse serum, 20 ng/ml epidermal growth factor (Merck CalBiochem, Darmstadt, Germany), 0.5 mg/ml hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA), 100ng/ml cholera toxin (Wako Pure Chemical, Osaka, Japan) and 10mg/ml insulin (Sigma-Aldrich). Human breast cancer, MCF-7 cells were obtained from Dr. Tabuchi in University of Toyama, and cells were cultured in MEM medium supplemented with 0.1 mM non-essential amino acids (Invitrogen, Carlsbad, CA, USA) and 10% fetal bovine serum. All cells were incubated in 5% CO2 humidified atmosphere at 37 C in culture media containing 100 U/ml penicillin G and 100mg/ml streptomycin (Invitrogen). Human recombinant TGF-b1 and TNF-a were purchased from PeproTech (Rocky Hill, NJ, USA). TGF-b type I receptor kinase inhibitor II (TGFRi) and Smad3 inhibitor (SIS3, 6, 7-dimethyl-2-[(2E)-3-(1-methyl-2-phenyl-1H-pyrrolo [2, 3-b] pyridin-3-yl-prop-2-enoyl)-1, 2, 3, 4-tetrahydroisoquinoline hydrochloride) were purchased from Merck CalBiochem.